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' . : . ~ Purpose
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To use peR to ampbfy DNA.
Practical peR
JJ Safety
If you undertake PCR make sure you are aware
of the hazards and foUow the instructions of
your teacher very carefully.
Scientists in forensics laboratories carry out the polymerase chain reaction (PCR) using
a machine called an automated thermal cycler. This is a programmable heating unit in
which the DNA to be amplified is incubated in a buffer solution with thermo-stable DNA
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polymerase, primers and deoxyribonucleotides. The unit maintains the cyclical sequence of
temperatures for the PCR process.
Your school or college may be lucky enough to possess a thermal cycler but it is possible to
carry out PCR without them, using three separate thermostatically controlled water baths.
You simply have to move the DNA sample from bath to bath - and complete 30 cycles! You
need a stopwatch, good teamwork and some sort of protection from the steam coming off
the hottest bath.
Having amplified the short tandem repeat sequences within your DNA sample, you will
then separate out the fragments using gel electrophoresis (see Practical 6.1). Comparing
the position of the bands on the gels to a standard or reference you will be able to draw
conclusions about the DNA sample you started with.
You will follow a practical protocol supplied by the company that produces the equipment
and reagents your school or college has purchased.
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Edexcel practical materials created by SaJters..Nuffield Advanced Biology, CUtrivenity of York Science EdUcatiOD Group.
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,
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. ~
, ~ ~ Purpose
~ Safety
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To use gel electrophoresis to separate DNA If you undertake gel electrophoresis make sure
you are aware of the hazards and follow the
instructions of your teacher very carefully.
fragments of different sizes.
Procedure
You may have the opportunity to complete experimental work using restriction enzymes and
gel electrophoresis or you may use the simulation of this.
Edexcd practical materials created by Salters-Nuffie1d Advanced Biology, ClUoiversity of York Science Education Group.
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When a bacterial infection is diagnosed it is useful to be able to tell to which antibiotics it is
most susceptible. In some cases this information is known, but in other cases tests need to
be carried out to find out which antibiotic will be most effective. In this activity you will be
testing the effectiveness of several types of antibiotics on bacteria.
The standard method of doing this is to put discs of chromatography blotting paper soaked
in the various antibiotics onto an agar plate that has been inoculated with the bacteria.
Alternatively a Mast ring (a ring of paper with several 'arms', each treated with a different
antibiotic) can be used.
Procedure
You will need:
Agar plate seeded with a known bacterium Marker pen
Bunsen burner Autoclaved forceps
Bench spray of disinfectant, 1% Virkon or Mast ring or antibiotic-impregnated paper
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equivalent discs
Soap or handwash Adhesive tape
Paper towels Eye protection
1 Wash your hands with the soap or handwash. Spray the working area thoroughly with the
disinfectant spray. Leave for at least 10 minutes, then wipe with a paper towel.
2 Work very close to a lit Bunsen burner. Prepare an agar plate seeded with bacteria. This
may have already been done for you. If not, follow the instructions in the section 'Pouring
agar plates' in Practical 4.3 EdexcelAS Biology. Label the Petri dish on the base at the
edge with your name, the date and the type of bacterium it is inoculated with.
3 Flame the forceps and then use them to pick up an antibiotic disc or Mast ring. Raise the
lid of the Petri dish and place the Mast ring firmly in the centre of the agar; if individual
discs are used they will need to be spaced evenly around the dish.
4 Tape the dish securely with two pieces of adhesive tape (but do not seal it completely),
then keep it upside down at room temperature for 48 hours.
Edexcel practical materials created by SaIters-Nuffidd Advanced Biology, OUoiversity of York Science Education Group.
, : ' : . ~ Purpose
".
To investigate the effect of different
antibiotics on bacteria.
Introduction
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o Safety
Wear eye protection.
The microorganisms are a potential biological hazard.
Use aseptic techniques when transferring the bacteria
to the Petri dishes. Clean the bench with antibacterial
disinfectant. Do NOT open the Petri dishes once they
have been incubated.
5 Wash your hands with soap or handwash and clean the bench again using the Virkon
spray.
6 After incubation, look carefully at the plate but do not open it. Where bacteria have
grown the plate will look opaque, but where the antibiotics have inhibited growth, clear
zones called inhibition zones will be seen. Measure the diameter of the inhibition zones
in millimetres and use this information to decide which antibiotic is most effective at
inhibiting the growth of the bacterium.
7 Collect data from other members of the class who used the other bacterial cultures.
8 Write a brief report of the results, comparing the different antibiotics and the effects on
the different bacterial cultures.
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Questions
1 Are the inhibition zones circular? If not, what is a sensible measuring strategy?
2 What factors determine the diameter of the inhibition zones?
3 If class data are shared:
a what is the overall spread of the data
b do all individual results show the same trends - if not, why not, and how could this
variability be represented on your graphs?
4 If you were working in a hospital laboratory, and you had just carried out this test on
bacteria isolated from sick patients, would you always choose the antibiotic that gave the
biggest inhibition zone? Are there any other factors you would need to consider?
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&!excel practical materials created by Salters-Nuffield Advanced Biology, CUniverstty of York Science Education Group.
HSW activity 1.3 Links lifestyle factors and heart ,disease .
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\"""7 ' urpose
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To interpret data on factors that increase the risk of heart disease.
To distinguish between cause and correlation,
To discuss how people use scientific knowledge to reduce their risk of coronary heart disease.
The following table shows a range of risk factors associated with coronary heart disease,
For each factor a best estimate and range of the percentage of heart disease attrIbutable
to that factor is given. (Note: the values given for cholesterol, blood pressure and fasting
glucose are 'beyond nonnal'.)
Risk factor Best estimate Range
blood cholesterol 20 mgldm3 or greater 43% 39-47%
physical inactivity 35% 23-46%
blood pressure 140/90 mm Hg or greater 25% 20-29%
cigarette smoking 2296 17-25%
obesity 1796 7-32%
diabetes (fasting glucose 14 mg/dm3 or greater) 896 1-15%
Source: Brownson RC, Remington PL, Davis jR, eds. Chrrmic Epidemiology rmd Control.
Washington DC: American Public H<!Qlth A6Iociation
Questions
1 Draw a graph or chart to show the data in the table.
2 From these data, which factors appear to have the greatest jrnpact on the risk of
developing heart disease? Comment on the reliability of your answer.
3 Give as many reasons as you can why the ranges vary so much,
4 What SOrt of evidence would you look for to determine whether the links between
these factors and heart disease are causal or just correlation?
S Most of the factors shown in the table are usually described as lifestyle factors.
Explain what we mean by a 'lifestyle factor' and identify which of the factors in the
table are lifestyle factors. Explain your answers,
6 Take one of the factors you identified as a lifestyle factOr and give a8 many reasons
as you can why, even though people know that it increases their risk of heart disease,
they do not change the way they live to decrease their risk.
&!excel pracdC2.1 materlals creoted by Safttn..Nuffield Biology, <:lUnh'::'!i:y == ..... '"